Biomedical Engineering Reference
In-Depth Information
lot. Besides, the separation of these biomolecules takes a different meaning than for
DNA where the problem is to discriminate between identical objects differing only
by their molecular weights. For proteins, completely different molecules differing
by their primary sequence and therefore their length, charge, hydrophilicity, and so
forth, have to be analyzed. The determination of the size is performed in Polyacryl-
amide gels in the presence of a surfactant, pH gradients are used to measure their
charge. Two-dimensional gels combine these two techniques.
10.1.5.1 SDS PAGE
This technique is used for separating proteins according to their size. As they are
globular charged objects taking various shapes, it is first necessary to unfold them.
This step is performed in a denaturating solution of sodium dodecyl sulfate (SDS).
This charged surfactant efficiently unfolds proteins so they can be considered as
classical flexible polyelectrolytes whose charge is imposed by the bounded SDS.
Since, on average, all the proteins have the ability to fix a similar amount of SDS
molecules (per unit length), all the proteins of the mixture can be approximated to
the same polyelectrolyte differing only in length even though their primary sequence
is different. Polyacrylamide gel electrophoresis (PAGE) of these SDS-protein com-
plexes is then conceptually similar to gel electrophoresis of DNA and is heavily used
particularly in the Ogston regime
m 
~
 
1/log(M) (10.18).
10.1.5.2 Isoelectric Focusing (IEF)
Because of the acidic and amine groups present in their structure proteins bear a
net positive charge at low pH and negative at high pH. The pH value at which
their charge is zero is called the isoelectric point. It is measured by creating a pH
gradient in a gel and by measuring the point where the net charge of the proteins
is strictly zero. When an electric field is applied to the gel, the mobility changes its
sign at this isoelectric point. A given protein is thus electrofocused and accumulates
at this point.
10.1.5.3 Two-Dimensional Electrophoresis
Electrophoresis can thus be used independently for molecular weight determination
or for measurement of the IEF. In these complex systems, it is common to combine
these two determinations by making 2-D electrophoresis (2-D PAGE) [31].
The principle is to make an IEF measurement over a pH gradient say along
the horizontal direction. This step sorts the proteins with respect to their charge.
The slab is then submitted to an SDS PAGE along the vertical axis that separates
all the proteins of the same spot (i.e., having the same charge) with respect to their
molecular weight (Figure 10.10).
These analyses are performed routinely and despite the complex patterns due
to the high diversity of the proteins of a particular sample, they give good and
reliable results for instance in following the evolution of the expression of a given
protein in different cell environments via the intensity of its related spot in the
electrophoregram.
